A comparative study of Sephadex, glass wool and Percoll separation techniques on sperm quality and IVF results for cryopreserved bovine semen

Abstract

The aim of this study was to compare the effects of spermatozoa separation techniques on sperm quality and in-vitro fertilization (IVF) results for cryopreserved bovine semen. Sephadex, glass wool and Percoll gradient separation techniques were used for sperm separation and sperm motility, morphology and membrane integrity were evaluated before and after separation. Also, cleavage and blastocyst developmental rate were investigated after IVF with sperm recovered by each separation technique. The motility of samples obtained by the three separation techniques were greater compared to the control samples (p < 0.05). The percentage of spermatozoa with intact plasma-membrane integrity, identified by 6-carboxyfluoresceindiacetate/propidium iodide fluorescent staining and the hypo-osmotic swelling test, was highest in the glass wool filtration samples (p < 0.05). The cleavage and blastocyst rate of total oocytes produced from glass wool filtration samples were also higher than the control and Sephadex filtration samples (p < 0.05), but were not significantly different from Percoll separation samples. However, a significantly greater number of cleaved embryos produced by glass wool filtration developed to blastocyst stage than those produced by Percoll separation (p < 0.05). These results indicate that spermatozoa with good quality can be achieved by these three separation techniques and can be used for bovine IVF. In particular, it suggests that glass wool filtration would be the most effective method of the three for improving sperm quality and embryo production for cryopreserved bovine spermatozoa.

Isolate® and Optiprep® minigradients as alternatives for sperm selection in bovine in vitro embryo production

“…However, no differences were detected in sperm kynetic and integrity of the acrosome and membrane in all treatment tested in this study. Many authors suggest that the sperm progressive motility is improved after Percoll centrifugation (Cesari et al 2006;Lee et al 2009;Machado et al 2009;Mehmood et al 2009;Petyim et al 2009). Similarly, progressive motility was improved, not only for P, but also for MP and MI.…”

Assessment of different in vitro sperm challenges and in vivo fertility of bovine semen batches

“…Several authors observed higher percentage of cells with intact plasma membrane in Percoll centrifuged semen (LEE et al, 2009;MACHADO et al, 2009;OLIVEIRA et al, 2012b), indicating that most dead spermatozoa present in frozen-thawed semen were retained in the upper layers of the gradient. Damaged spermatozoa are removed by a particular mechanism in Percoll separation methods (OSHIO, 1988;RHEMREV et al, 1989) and sperm progressive motility is generally improved after Percoll centrifugation (CESARI et al, 2006;LEE et al, 2009;MACHADO et al, 2009;PETYIM et al, 2009). In the present study the small amount of semen (150 µL) being deposited in the upper layer of this density gradient might have avoided this phenomenon to happen.…”

“…Table 3 -Mean (± SD) of plasma membrane integrity (PMI; %), sperm morphology (major, minor and total defects; %), sperm concentration (CONC; x10 6 sptz/mL), and number of sperm presenting membrane integrity (PMI*CONC; x10 6 sptz/ mL) after different laboratory challenges using different batches from a Brangus bull -Sertãozinho, SP -January 2016 Percoll centrifugation is a widely used method for bovine sperm selection (MCCANN;CHANTLER, 2000;PETYIM et al, 2009) mainly before IVP (CESARI et al, 2006;LEE et al, 2009;MACHADO et al, 2009). Several authors observed higher percentage of cells with intact plasma membrane in Percoll centrifuged semen (LEE et al, 2009;MACHADO et al, 2009;OLIVEIRA et al, 2012b), indicating that most dead spermatozoa present in frozen-thawed semen were retained in the upper layers of the gradient. Damaged spermatozoa are removed by a particular mechanism in Percoll separation methods (OSHIO, 1988;RHEMREV et al, 1989) and sperm progressive motility is generally improved after Percoll centrifugation (CESARI et al, 2006;LEE et al, 2009;MACHADO et al, 2009;PETYIM et al, 2009).…”

The Effect of Semen Washing and Soybean Lecithin Level on Motility and Viability of Ram Spermatozoa Stored at 5⁰c

“…The sperm quality after semen washing (Table 2) was also still in good condition for being further processed. The success of semen washing process have been reported by many researchers with a variety of methods (Somfai et al,2002;Councel et al, 2004;Lee et al, 2009;Marcus, 2010).…”